We have continued our analyses of mutant Chinese hamster ovary cells pleiotrophically defective in receptor-mediated endocytosis. Based on similarities observed between the mutants and normal cells treated with agents that dissipate pH gradients, we hypothesized that these mutants were unable to present internalized ligands to an acidic compartment. Endosomes isolated from mutant DTG-1-5-4 showed no ATP-dependent acidification, suggesting that this mutant may have a defective proton pump. Endosomes from a phenotypically similar mutant, DTF 1-5-1, exhibited normal acidification. In contrast, transferrin was found to cycle through DTF 1-5-1 without releasing bound iron, a reaction that depends on acidification of the endosomes. Hybrids of DTF 1-5-1 X DTG 1-5-4 showed genetic complementation, thus the lesions in these mutants lie in different genes. The defect in DTF 1-5-1 has yet to be identified. Release of Sindbis virions from DTF 1-5-1 and DTG 1-5-4 was decreased (20 and 4% of normal, respectively) under conditions of infection such that viral proteins were synthesized at normal levels. In this respect also the mutants resemble cells in which maintenance of pH and ion gradients is inhibited. Comparison of viral glycoproteins isolated from mutant and parental cells indicate that some step(s) in post-translational modification is omitted in the mutants. By radiolabeling with various metabolites we have shown that initial glycosylation, processing of the oligosaccharide through fucosylation, acylation and proteolytic cleavage of the viral glycoproteins all occur in the mutants. Also, at least one of the glycoproteins is transported to the plasma membrane. Through biochemical, morphological and genetic analyses of mutants such as these, we are attempting to dissect the complex pathways of endocytosis and secretion and to establish the points of intersection between these pathways.